Optimised formulation of tobramycin for aerosolization

ABSTRACT

The invention provides a tobramycin formulation for delivery by aerosolization in the form of additive-free, isotonic solution whose pH has been optimised to ensure adequate shelf-life at room temperature. Said formulation can be advantageously used for the treatment and prophylaxis of acute and chronic endobronchial infections, in particular those caused by the bacterium  Pseudomonas aeruginosa  associated to lung diseases such as cystic fibrosis.

[0001] The present invention relates to tobramycin formulations fordelivery by aerosolization.

SUMMARY OF THE INVENTION

[0002] The invention provides a tobramycin formulation for delivery byaerosolization in the form of additive-free, isotonic solution whose pHhas been optimised to ensure adequate shelf-life at room temperature.

[0003] Said formulation can be advantageously used for the treatment andprophylaxis of acute and chronic endobronchial infections, in particularthose caused by the bacterium Pseudomonas aeruginosa associated to lungdiseases such as cystic fibrosis.

PRIOR ART

[0004] Although pressurised metered dose inhalers (MDIs) and dry powderinhalers (DPIs) are the most commonly used inhalation drug deliverysystems, nebulisers have become increasingly popular for the treatmentof airway obstruction, particularly in young children with asthma and inpatients with severe asthma or chronic airflow obstruction. Nebulisersuse ultrasound or compressed gas to produce aerosol droplets in therespirable size range (1- to 5 μm) from liquids, usually aqueoussolutions or suspensions of drugs. They have the advantage over MDIs andDPIs that the drug may be inhaled during normal breathing through amouth-piece or a face-mask. Thus, they can be employed to deliveraerosolised drug to patients, such as children, who experiencedifficulties using other devices.

[0005] Several types of therapeutically useful drug can be delivered bynebulisers, including β2-agonists, corticosteroids, anticholinergics,anti-allergics, mucolytics and antibiotics. The major clinical settingin which therapy with aerosolised antibiotics has been tried is themanagement of patients with cystic fibrosis (CF).

[0006] CF is a common genetic disease that is characterised by theinflammation and progressive destruction of lung tissue. Thedebilitation of the lungs in CF patients is associated with accumulationof purulent sputum produced as a result of endobronchial infectionscaused in particular by Pseudomonas aeruginosa. The latter ones are amajor cause of morbidity and mortality among patients with CF.

[0007] Tobramycin is an aminoglycoside antibiotic specifically activeagainst Pseudomonas aeruginosa. It penetrates endobronchial secretions(sputum) poorly, necessitating large intravenous doses to attain anefficacious concentration at the site of infection. These high dosesplace the patient at risk for nephrotoxic and ototoxic effects. Thedirect delivery of tobramycin to the lower airways by aerosoladministration is attractive, since it produces high concentrations ofantibiotic at the site of infection. In view of the limited absorptioninto the circulation, aerosol delivery of tobramycin should beassociated with minimal systemic toxicity. This would allow for thedevelopment of a safer, long-term therapy.

[0008] At this regard, being its therapeutic dose quite large,nebulisation turns out to be extremely convenient due to theimpossibility of formulating tobramycin into an MDI or DPI.

[0009] The clinical studies reported in the literature showcontradictory results in terms of benefit from aerosolised tobramycin inpatients with CF. The variability among these studies might, in part,result from the differences in the patient population, therapeuticmodalities, nebulisers, formulations and their mode of administration.Furthermore, most of the studies have been carried out through theextemporaneous use of the commercially available injectable solutions.These preparations normally contain anti-oxidant and preservatives whichare known to cause paradoxical reactions such as bronchospasm and cough(Nikolaizik et al Eur J Pediatr 1996, 155, 608-611; The Lancet, Jul.23^(rd) 1988, 202).

[0010] For all these reasons, there is a need for standardisedprocedures as well as for improvement in aerosol administration ofantibiotic such as tobramycin to CF patients.

[0011] Therefore, in consideration of all problems outlined, it would behighly advantageous to provide a tobramycin formulation of atherapeutically useful concentration deliverable by aerosolization intothe endobronchial space which: i) could be efficiently nebulised in arelatively short time using both jet and ultrasonic nebulisers; ii)could permit generation of aerosol well-tolerated by patients; iii) isable to produce aerosol particles which can efficaciously reach thetherapeutic target area; iv) is rid of substances (preservatives andothers) that may give rise to undesirable side effects; v) couldguarantee as long as possible a shelf-life, in particular at roomtemperature.

[0012] Accordingly, in order to obtain an optimised formulation fortobramycin aerosol administration, the following parameters need to becarefully adjusted:

[0013] The ratio dose/volume. Formulation for aerosol delivery shouldcontain the minimal yet efficacious amount of tobramycin formulated inthe smallest as possible volume of solution. In fact, the smallest thevolume, the shortest the nebulisation time. A short nebulisation time,in turn, is an important determinant of patient compliance and withinhospitals has implications for staff time (McCallion et al Int J Pharm1996, 130, 1-11).

[0014] The osmolarity. It is well known that adverse reactions toinhalation therapy may be caused by hypo- or hyper-osmolarity of drugsolutions. On the contrary, isotonic solutions remove the risk ofparadoxical bronchoconstriction and cough (The Lancet, 1988, op. cit.;Mann et al Br Med J 1984, 289, 469). The osmolarity also affects theperformances of the nebulisers in terms of output rate and particle sizedistribution (vide ultra).

[0015] The particle size distribution upon nebulisation. The efficacy ofa clinical aerosol is dependent on its ability to penetrate therespiratory tract. To penetrate to the peripheral regions, aerosolsrequire a size from 0.8 to 5 μm, with a size of about 3 μm preferablefor alveolar deposition. Particles smaller than 0.5 μm are mainlyexhaled. Besides the therapeutic purposes, the size of aerosol particlesis important in respect to the side effects of the drugs. Largerdroplets deposited in the upper respiratory tract are indeed rapidlycleared from the lung by the mucociliary clearance process, with theeffect that drug becomes available for systemic absorption andpotentially adverse effects. The same problems could occur with toosmall aerosol particles which, due to deep lung penetration, might giverise to higher systemic exposure so enhancing the undesired systemiceffects of the drugs. Several authors (Newman et al Thorax 1988, 43,318-322; Smaldone et al J Aerosol med, 1988, 1, 113-126; Thomas et al.Eur Respir J 1991, 4, 616-622) have suggested that close attention tothe droplet size of the aerosolised drug for the antibiotic treatment ofCF must be paid, since penetration to the peripheral airways isparticularly desirable.

[0016] The pH of the formulation. An important requirement for anacceptable formulation is its adequate shelf-life suitable forcommercial, distribution, storage and use. Generally, tobramycinintravenous solutions contain phenol or other preservatives andanti-oxidants to maintain potency and to minimise the formation ofdegradation products that may colour the solution. However, as alreadypointed out, said substances may induce unwanted reactions in patientswith lung diseases such as CF. The stability of tobramycin strictlydepends on the pH. Therefore, the pH of its formulations need to becarefully adjusted in order to slow or prevent degradation productsformation without the aid of preservatives and/or anti-oxidants; itwould also be advantageous to adjust pH in such a way as to prevent asmuch as possible discoloration although the depth of colour is not areliable indicator of the extent of oxidation. Formulations provided ofadequate shelf-life under environmental storage conditions (roomtemperature and, at the occurrence, protected form light) would beparticularly preferred, since the stability at room temperature of thepreparations of the prior art are rather unsatisfactory. During use, theformulation prepared according to EP 734249 marketed under thetrade-name of Tobi® could be indeed kept at room temperature for only 28days.

OBJECT OF THE INVENTION

[0017] It is an object of the invention to provide a formulation to beadministered by nebulisation suitable for well-tolerated and efficaciousdelivery of tobramycin into the endobronchial space for treatingPseudomonas aeruginosa and/or other susceptible bacterial infectionsassociated to pulmonary diseases such as CF.

[0018] In particular, it is an object of the invention to provide aformulation in the form of aqueous solution to be administered bynebulisation, wherein tobramycin concentration, tonicity and pH havebeen optimised for guaranteeing better compliance of the patients,maximal tolerance and efficacy and as long as possible a shelf-life atroom temperature.

[0019] According to the present invention there is, provided aformulation constituted of 7.5% w/v tobramycin in an aqueous solutionhaving a pH of between 4.0. and 5.5 and osmolarity between 250 and 450mOsm/l (approximately equivalent to mOsm/kg).

[0020] In a preferred embodiment of the invention, the formulationcontains 300 mg of tobramycin sulfate in 4 ml of half-saline aqueoussolution (0.45% of sodium chloride) in order to have an osmolarityranging from 280 to 350 mOsm/l and it has a pH of 5.2.

[0021] In the prior art, several tobramycin formulations for inhalationhave been proposed for the treatment of patients with CF and Pseudomonasaeruginosa infections.

[0022] Most of the commercially available tobramycin solution forinjection when extemporarily used for inhalation can cause significantbronchial obstruction as they are not preservative-free but containanti-oxidants such as sodium EDTA and/or sodium metabisulphite andpreservatives such as phenol.

[0023] Wall et al (The Lancet, 1983, Jun. 11^(th), 1325) reported theresult of a clinical study upon inhalation of 80 mg tobramycin plus 1 gticarcillin twice daily from a hand-held nebuliser. On their ownadmission, one of the drawbacks of the regimen is the time required forinhalation (about 30 min).

[0024] Ramsey et al (New Eng J Med 1993, 328, 1740-1746) conducted anextensive study to evaluate the safety and efficacy of aerosolisedtobramycin. For reaching the target concentration (≧400 μg per gram ofsputum), they used 600 mg of preservative-free tobramycin sulfatedissolved in 30 ml of half-strength physiologic saline, adjusted to a pHof 6.85 to 7.05. The large volume was required by the ultrasonicnebuliser used (DeVilbiss). Besides the long term required forinhalation, the pH is not optimal either. From a stability point ofview, it is known that, at pH around neutrality, tobramycin rapidlyoxidises although it is very stable towards hydrolysis (Brandl et alDrug Dev Ind Pharm 1992, 18, 1423-1436). Common Compendia (Martindale,Physician Desk Reference) suggest indeed to maintain tobramycin solutionat a pH comprised between 3.0 and 6.5.

[0025] EP 734249 claims a formulation comprising from 200 mg to 400 mgof aminoglycoside dissolved in about 5 ml of solution containing 0.225%of sodium chloride (¼ normal saline—NS-) and having pH between 5.5 and6.5. According to the inventors, the formulation contains minimal yetefficacious amount of aminoglycoside formulated in a small as possible avolume of physiologically acceptable solution having a salinity adjustedto permit generation of aminoglycoside aerosol well-tolerated bypatients but preventing the development of secondary undesirable effectssuch as bronchospasm and cough (pg. 4, lines 51-55). The preferredtobramycin formulation containing ¼ NS with 60 mg of tobramycin per mlof ¼ NS (which equates to 6% w/v) has a pH of about 6.0 and anosmolarity in the range of 165-190 mOsm/l. According to the inventors,the osmolarity range is within the safe range of aerosols administeredto a cystic fibrosis patient and a further advantage of the quarternormal saline, i.e. saline containing 0.225% of sodium chloride with 60mg/ml tobramycin is that this formulation is more efficiently nebulisedby an ultrasonic nebuliser compared to tobramycin formulated in asolution 0.9% normal saline (pg. 5, lines 50-54). The inventors statethat a more concentrated solution (in comparison to 60 mg per ml) willincrease the osmolarity of the solution, thus decreasing the output ofthe formulation with both jet and ultrasonic nebulisers. Alternatively,a more concentrated solution in a smaller volume is also disadvantageousdue to the typical dead space volume of the nebulisers (1 ml): thatmeans that the last 1 ml of solution is wasted because the nebuliser isnot fully performing (pg. 6, lines 35-38). The claimed pH range wasfound to be optimal from the storage and longer shelf-life point of view(page 7 lines 2-3) but, indeed, it allows to achieve completely stablesolutions at 5° C. and effectively stable ones at room temperature for 6months; moreover the claimed formulation remain within an acceptablerange of color obtained upon storage in pouch (so protected from light),but there is no data referring to its behaviour outside the pouch.

[0026] A pH between 5.5 and 6.5 was claimed because, in the Opinion ofthe inventors, any aerosol with a pH of less than 4.5 usually willinduce bronchospasm in a susceptible individual and aerosols with a pHbetween 4.5 and 5.5 will occasionally cause this problem (pg. 5, line58-pg. 6, line 1) Le Brun et al Int J Pharm 1999, 189, 205-214 discloseda 10% w/v tobramycin solution for inhalation having a pH of 7.5. Thesame authors (Int J Pharm 1999, 189, 215-225), in a further study aimingat developing highly concentrated solutions, have studied theaerosolization properties of several tobramycin solutions, ranging from5 to 30% w/v. All the solutions disclosed in this paper have a pH aroundthe neutrality and exhibit an osmolarity far away from an isotonic value(282 mOsm/l).

[0027] In none of aforementioned documents the features of theformulation of the present invention are disclosed and none of theteaching therein disclosed fully contributes to the solution of theproblem underlying the invention, to provide a concentrated solution tobe delivered by aerosol in a smaller volume, with a tonicity closer tothe physiological value.

[0028] The use of a more concentrated solution with respect to thatreported as optimal in the prior art (7.5% vs. 6.0% w/v) allows toemploy vials with a smaller volume, so allowing, in turn, to reduce thetime of nebulisation. Although it is true that some nebulisers have adead space volume of 1 ml, other have a minor one (0.5 ml or less), sothe wasting of using vials of 4 ml would be only approx. 10% or less.

[0029] According to the invention, the osmolarity of the formulation iswithin the range of solutions considered as isotonic, whereas both theformulations of EP 734249 and Ramsey et al have an osmolarity, i.e.165-190 mOsm/l, typical of solutions considered as hypotonic (Derbracheret al Atemwegs und Lung 1994, 20, 381-382). Although the formulations ofthe prior art turned out to be safe, only isotonic solutions maycompletely prevent the risk of paradoxical bronchoconstriction.Moreover, the results reported in the example 2 indicate that theformulations having an osmolarity in the range claimed, contrary to whatstated in EP 734249, are efficiently nebulised despite their higherconcentration.

[0030] The pH between 4.0 and 5.5, preferably 5.2, was found to beoptimal in terms of storage and shelf-life at room temperature.Long-term stability studies show that tobramycin in the formulation ofthe present invention is stable for over nine months. Moreover, for allthat period, its colour does not significantly change and remains withinan acceptable range even if not stored in a foil overpouch.

[0031] According to a further embodiment of the invention, there is alsoprovided a process for the preparation of such formulation, said processincluding the steps of:

[0032] i) preparing an aqueous solution containing 0.45% w/v of sodiumchloride;

[0033] ii) adjusting the pH with a concentrated strong acid;

[0034] iii) adding the active ingredient and mixing to completedissolution;

[0035] iv) re-adjusting the pH to the desired value;

[0036] v) filling the solution in suitable containers, preferablypre-sterilised by filtration.

[0037] The aerosol formulations of the invention refer to a 7.5% w/vaqueous solution of an antibiotic of the aminoglycoside family,preferably tobramycin and salts thereof, for the treatment of lunginfections due to Gram positive and negative bacteria, in pulmonarydiseases such as cystic fibrosis, non-CF bronchiectasis infected withPseudomonas aeruginosa and other chronic pneumopathies, particularly inan exacerbation phase, such as bronchiectasis, COPD and bronchialasthma.

[0038] The osmolarity of the formulation should range between 250 and450 mOsm/l, preferably between 260 and 400, even more preferably between280 and 350 mOsm/l; it can be adjusted by using any physiologicallyacceptable salt or non-volatile compounds; preferably, tobramycin isdissolved in a 0.45% sodium chloride aqueous solution.

[0039] The pH can be adjusted by using any concentrated strong acid,preferably sulfuric acid and should range from 4.0 to 5.5, preferablyfrom 5.0 to 5.4.

[0040] The formulations of the invention can be distributed in suitablecontainers such as multidose vials or pre-sterilised unit dose vials of2 or 4 ml, depending on the therapeutic indication; otherwise, the vialscan be aseptically filled using the “blow, fill and seal” technology.The filling is preferably carried out under inert atmosphere. Thesolution formulations can be advantageously sterilised by filtration.

[0041] The invention is illustrated by the following examples.

EXAMPLE 1 Preparation of the 7.5% w/v Tobramycin Solution at pH 5.2 andStability Studies

[0042] The composition refers to 1 unit-dose vial (2 ml) IngredientQuantity Tobramycin 150 mg Sodium chloride  9 mg Sulphuric Acid 2N q.s.to pH 5.2 ± 0.2 Sodium hydroxide 1M* q.s to pH 5.2 ± 0.2 Purified waterq.s. to 2 ml

[0043] Sodium chloride is dissolved into 40 l of purified water (mix for15 minutes to guarantee the NaCl complete dissolution). Then, 30 l ofsulphuric acid (2N H₂SO₄) is added to the saline solution; during theoperation, the solution temperature is monitored. When the solutiontemperature is about 25÷30° C., N₂ is insufflated to obtain a value ofdissolved O₂ less than 1 mg/l. Afterwards, tobramycin is added and mixedto complete dissolution (for not less than 15 minutes) while thetemperature is maintained below 25-30° C. The pH value is checked and,if necessary, sulphuric acid 2N or sodium hydroxide solution 1M areadded to obtain a pH value of 5.2±0.2. When the solution temperature is25° C.±2° C., purified water is added to reach the final volume. Theresulting solution is mixed for 15 minutes. The pH value is checkedagain and, if necessary, sulphuric acid 2N or sodium hydroxide solution1M are added to obtain a pH value of 5.2±0.2. The solution is filteredthrough one 0.45 μm Nylon filter, and through two 0.2 μm Nylon filters.

[0044] The solution is distributed in 2 ml polyethylene colorless unitdose vials under nitrogen purging.

[0045] The stability of the vials was evaluated both under long-term(25° C., 60% R.H.) and accelerated conditions (40° C., 75% R.H.)[R.H.=relative humidity]. Results are reported in Tables 1 and 2,respectively. Assays of tobramycin and of its main related substances(degradation products) were determined by HPLC. Residual oxygen, pH andosmolarity were also assayed. The osmolarity was measured using afreezing-point depression osmometer.

[0046] The formulation of the invention turns out to be stable for atleast 9 months at room temperature and for 6 months under acceleratedconditions. pH and osmolarity remain substantially unchanged under bothconditions. At room temperature, the colour of the formulation of theinvention does not significantly change and remains within an acceptablerange even if not stored in a foil overpouch. TABLE 1 Stability underlong-term conditions (25° C., 60% R.H.) TECHNOLOGICAL CONTROLS CHEMICALCONTROLS Solution Oxygen Osmolarity Tobramyicin Related Analysisappearance mg/l mOsm/l mg/ml (%) pH substances % Specification Clearpale (a) 260-350 67.5-82.5 4.5-5.5 (a) range yellow solution t = 0 Clearpale 2.8 321 75.8 (100) 5.2 6.39 yellow solution t = 3 months Clear pale2.9 314 76.6 (101.1) 5.2 6.35 yellow solution t = 6 months Clear pale3.0 304 75.4 (99.5) 5.0 5.84 yellow solution t = 9 months Clear pale 2.8293 76.5 (100.9) 5.1 6.27 yellow solution

[0047] TABLE 2 Solution of example 1 - Stability under acceleratedconditions (40° C., 75% R.H.) TECHNOLOGICAL CONTROLS CHEMICAL CONTROLSSolution Oxygen Osmolarity Tobramycin Related Analysis appearance mg/lmOsm/l mg/ml (%) pH substances % Specification Clear pale (a) 260-35067.5-82.5 4.5-5.5 (a) range yellow solution t = 0 Clear pale 2.8 32175.8 (100) 5.2 6.39 yellow solution t = 1 months Clear pale 2.7 n.d.78.3 (103.3) 5.1 6.42 yellow solution t = 3 months Clear 2.8 311 75.3(99.3) 5.1 5.48 yellow solution t = 6 months Clear 3.2 292 77.1 (101.7)4.7 6.17 yellow solution

EXAMPLE 2

[0048] The nebulisation efficiency of the solution for inhalation ofexample 1, expressed as percentage of active ingredient nebulised, wasevaluated using a commercial jet nebuliser (PARI-BOY) for a 5-minutenebulisation time. The size profile of the droplets produced bynebulisation of the solution, expressed as diameter (μm) below whichrespectively 10%, 50% and 90% of the droplets are included, was alsocharacterised by Malvern analysis.

[0049] A formulation prepared according to the teaching of the preferredembodiment of EP 734249, i.e. containing 60 mg of active ingredient perml of 0.225% sodium chloride aqueous solution and having a pH of about6, was nebulised for comparison. Both formulations were filled in 2 mlunit dose vials. The results are reported in Table 3 as a mean of twodeterminations. TABLE 3 Malvern Analysis Osmolarity (μm) Efficiency(mOsm/l) 10% 50% 90% (%) Formulation 295 1.61 6.26 13.46 47.4 of ex. 1Formulation 222 1.78 6.24 13.46 42.2 of EP 734249

[0050] The results indicate that the formulation according to example 1having an osmolarity near isotonicity is efficiently nebulised.

[0051] The size profile of the droplets produced by nebulisation isinstead practically the same.

1. An aerosol formulation consisting of 75 mg/ml of tobramycin dissolvedin a 0.45% sodium chloride wherein the pH is comprised between 4.0 and5.5 and the osmolarity ranges between 250 and 450 mOsm/l.
 2. An aerosolformulation according to claim 1 wherein the pH is 5.2 and theosmolarity is between 280 and 350 mOsm/l.
 3. A process for thepreparation of an aerosol formulation according to claim 1, said processincluding the steps of: i) preparing an aqueous solution containing0.45% w/v of sodium chloride; ii) adjusting the pH with a concentratedstrong acid; iii) adding the active ingredient and mixing to completedissolution; iv) re-adjusting the pH to the desired value; v) fillingthe solution in suitable containers, preferably pre-sterilised byfiltration.